In gel analysis and sodium-dodecyl sulfate denaturation, AKAP12 behaved
with a MW of a homo-dimer. Only in the presence of the chaotropic agent
8 M urea did gel analysis reveal a monomeric form of AKAP12. By
separation by steric-exclusion chromatography, AKAP12 migrates with MW
of ~840 kDa, suggestive of higher-order complexes such as a tetramer.
Interestingly, the N-(1-840) and C-(840-1782) terminal regions of AKAP12
themselves retained the ability to form dimers, suggesting that the
structural basis for the dimerization is not restricted to a single
"domain" found within the molecule. In either sodium dodecyl sulfate or
urea, AKAP5 displayed a relative mobility of a monomer, but by
co-immunoprecipitation in native state was shown to oligomerize. When
subjected to steric-exclusion chromatography, AKAP5 forms higher-order
complexes with MW ~220 kDa, suggestive of tetrameric assemblies.

Conclusion

Both AKAP5 and AKAP12 display the capacity to form supermolecular
homo-oligomeric structures that likely influence the localization and
function of these molecular scaffolds.

Background

Discovery of a docking site for the regulatory subunits (i.e.,
RI/RII) of cyclic AMP-dependent protein kinase A (PKA, A-kinase) was seminal in
our understanding of the roles of A-kinase-anchoring protein (AKAP) scaffolds in
cellular signaling [1-4]. AKAPs not only dock PKA, but act as
molecular "tool boxes" that are multivalent and capable of docking other protein
kinases (protein kinase C and the tyrosine kinase Src), phosphoprotein
phosphatases, such as protein phosphatase 2B (PP2B) [5], cyclic AMP phosphodiesterases (PDE) [6-9],
adaptor molecules [8-11], ion channels [12-14], and members of the
superfamily of G protein-coupled receptors (GPCR) [15-17]. Two AKAPs,
AKAP5 and AKAP12, that associate with the prototypic GPCR, the
β2-adrenergic receptor (β2-AR), have been
the focus of intense research [16,18-21]. Herein, we examine these two members of the class of
GPCR-associated AKAPs and explore the extent to which these proteins, whose
structure is predicted to be natively unordered [5], are capable of forming oligomers. The current work is the first
to report that both AKAP5 and AKAP12 are not only capable of forming homodimers,
but also of forming higher-order supermolecular homo-oligomeric complexes. Thus
AKAP oligomerization adds a new dimension on how members of this class of
scaffold molecules operate in cell signaling.

ResultsOligomerization of AKAP12

We first sought to interrogate if oligomerization of AKAP12 could be detected
using purified human AKAP12. Consequently, His-tagged human AKAP12 was
expressed in E. coli, purified by gel filtration
chromatography, and analyzed by SDS-PAGE. Analysis of the expressed,
purified AKAP12 in the presence of the denaturant SDS revealed a
Mr of ~500 kDa, slightly more than twice
the mass of that calculated (hAKAP12, MW = 191.5 kDa) based upon primary
sequence alone (see protein staining, Figure 1A). The nature of the 500 kDa-Mr
species was made clear by performing the same analysis, but
with prior sample treatment and SDS-PAGE separation performed in the
presence of the chaotropic agent 8 M urea (Figure 1B). In the presence of 8 M urea, the apparent
Mr of purified AKAP12 declined from ~500
kDa to ~250 kDa, emblematic of a monomeric form of this large AKAP. The
results obtained of fixed gels, stained for protein (Figure 1A, B) were further tested using
immunoblotting to detect the protein (Figure 1C, D). Immunoblots of the resolved, transferred protein that
were stained with antibodies to the His-tag confirmed the results of the
protein staining (Figures 1C, D).
AKAP12 forms oligomers that are SDS-resistant. Homodimeric AKAP12 can only
be disassembled by treatment and separation in 8 M urea, a strong chaotropic
agent.

Figure 1

Oligomerization of AKAP12 (and either C-terminal or N-terminal
fragments) expressed and purified from E.
coli. His-tagged AKAP12 or His-tagged AKAP12
fragments were expressed in E. coli. The AKAP12 (or
fragments) were purified and then resolved on SDS-PAGE in the
absence (A) or presence of 8 M urea (B). The gels were fixed and
stained for protein with Coomassie Brilliant Blue (A, B) or the
resolved proteins transferred to PVDF membrane and subjected to
immunoblotting (C, D). The "◀" indicates the observed
Mr on the gels and the "Δ"
indicates the predicted Mr, calculated
from the primary sequence.

Since we established the existence of AKAP12 oligomers using SDS-PAGE, we
probed the upper limit size of AKAP12 oligomers. AKAP12 purified from
E. coli was subjected to steric-exclusion
chromatography on a high-resolution, wide-bore matrix operated by an AKTA
FPLC system (Figure 2). A spark
symmetrical peak of protein (A280) and of AKAP12 immunoreactivity (see blot
inset) was resolved. Using a set of marker proteins of known
Mr, we were able to size the AKAP12
supermolecular complexes with precision. The Mr
of the AKAP12 was 830-850 kDa. AKAP12 appears to form
oligomers greater than dimers, minimally trimeric or even tetrameric. This
higher order assembly of AKAPs into supermolecular complexes is a novel
observation.

Figure 2

Steric-exclusion chromatography of purified AKAP12.
His-tagged AKAP12 was purified and then resolved by steric-exclusion
chromatography performed on an AKTA FPLC fitted with HiPrep
Sephacryl-S400 (16/60) column. The presence of supermolecular
oligomers of AKAP12 was established by SDS-PAGE and immunoblotting
of samples from the chromatography. The resolved, transferred
proteins were stained with anti-AKAP12 antibodies. Marker protein
mobilities were employed to establish the Mr
of AKAP12 by elution position (inset). The
results displayed are representative of three separate experiments
performed on as many separate cell cultures.

We probed the same queries for AKAP12 expressed in mammalian cells. Human
embryonic kidney 293 (HEK293) cells transfected with an expression vector
harboring the human AKAP12 were collected, lysed, and the mobility of AKAP12
established by SDS-PAGE (Figure 3A).
Homodimers of HA-tagged AKAP12 exogenously expressed in HEK293 cells were
found to be resistant to SDS denaturation. By SDS-PAGE analysis, a ~500
kDa-Mr immunoreactive band was obvious,
whether the immunoblots were stained with anti-AKAP12 or anti-HA antibodies
(Figure 3A). When the samples were
treated with and subjected to SDS-PAGE in the presence of 8 M urea, the
Mr of the immunoreactive band of AKAP12
declined from ~500 kDa to ~250 kDa (Figure 3B). This ~250 kDa mobility is consistent with the MW calculated
for the monomeric form of the scaffold protein from primary sequence.

Figure 3

Oligomerization of AKAP12 (and either C-terminal or N-terminal
fragments) expressed in mammalian HEK293 cells. Human
embryonic kidney (HEK) 293 cells were transfected with an empty
expression vector or one harboring either full-length AKAP12, AKAP12
N-terminal (1-938) or C-terminal (840-1782) regions. Whole-cell
lysates of cells expressing either AKAP12 or one of the fragments
were subjected to SDS-PAGE in the absence (A) or presence of 8 M
urea (B). The resolved proteins were transferred to PVDF membrane
and subjected to immunoblotting, stained for AKAP12 or the HA-tag.
The results displayed are representative of at least three separate
experiments performed on as many separate cell cultures.

We investigated if N-terminal (1-840) and C-terminal (840-1782) fragments of
AKAP12 would retain the ability to form dimers, as observed with the
full-length AKAP12 (Figure 1A, B).
Expression vectors harboring either the His-tagged N-terminal or the
C-terminal region of AKAP12 were constructed, the protein was expressed in
E. coli, and each of the two large fragments was
purified by affinity chromatography. Protein staining of the N- and
C-terminal fragments resolved by gel analysis in the presence of SDS
reveals, remarkably, the homodimerization of both the N-terminal as well as
the C-terminal fragments of AKAP12 (Figure 1A). In the presence of 8 M urea, however, the N-terminal and
C-terminal fragments of AKAP12 now can be resolved as monomeric forms
(Figure 1B). Immunoblotting of the
His-tagged fragments resolved in SDS (Figure 1C, D) confirms the identity of the species made visible by
protein staining (Figure 1A, B),
i.e., both AKAP12 fragments behave as dimers in
SDS-PAGE. In the presence of 8 M urea, the N- and C-terminal fragments made
visible by protein staining (Figure 1B)
likewise resolve as the monomeric forms, as made visible by immunoblotting
(Figure 1C, D).

The electrophoretic behavior of full-length human AKAP12, as well as the N-
and the C-fragments was probed when these fragments were expressed in
mammalian (HEK293) cells, rather than in E. coli. The
expressed HA-tagged versions of the full-length AKAP12 (1-1782), the
N-terminal fragment (1-938), and a C-terminal (840-1782) fragment of AKAP12
were subjected to SDS-PAGE, immunoblotting of the resolved proteins, and
made visible by staining with anti-HA antibody (Figure 3A, B). In the presence of SDS, HA-tagged AKAP12
displays a Mr of a dimer (Figure 3A). Immunostaining of the HEK cell
extracts with anti-AKAP12 antibody likewise displays the same homodimeric
character. When treated and resolved on gels in the presence of 8 M urea,
either exogenously (probed with anti-HA immunostaining) or endogenously
(probed with anti-AKAP12 antibody) expressed AKAP12 migrated with a
Mr of a monomer (Figure 3B). Both the N-terminal as well as the
C-terminal fragments of AKAP12 expressed in mammalian cells displayed dimers
in the presence of SDS (Figures 1A,
3A) and monomers in the presence of
8 M urea (Figures 1B, 3B). Thus we establish that the dimeric
character of AKAP12 and these two large AKAP fragments reflects neither the
presence/absence of tags nor whether the proteins are exogenously expressed
in E. coli or endogenously in mammalian cells, i.e., native
AKAP12 oligomerizes.

Since AKAP12 mediates recycling and resensitization of beta-adrenergic
receptors [4,5,22-26], which mediate elevation of
intracellular cyclic AMP, we sought to interrogate the effects of modulating
cyclic AMP signaling pathway on the oligomerization of AKAP12 (Figure 4). Stimulation of cyclic AMP
accumulation in HEK293 cells by stimulation of the cells with
beta-adrenergic agonist, isoproterenol, for 5 or 30 min had little effect on
the mobility of AKAP12 on SDS-PAGE, ~500 kDa-Mr
(Figure 4A). To test
this possibility further, we examined the effect of mutating the three sites
of protein kinase A-catalyzed phosphorylation of AKAP12 on the ability of
the AKAP12 to form oligomers [22].
AKAP12 mutated by alanine substitution of protein kinase A phosphorylation
sites (Ser627Ala, Ser696-698Ala, Ser772Ala, labeled "AKAP12M3") was
expressed in HEK293 cells, the cells treated without or with isoproterenol,
and the Mr of AKAP12 established by SDS-PAGE
(Figure 4A). AKAP12M3 displayed the
same Mr (~500 kDa) as native AKAP12, i.e., no
change in dimer formation in response to stimulation of the cells with
isoproterenol. Cells were then challenged with inhibitors to protein kinase
A (KT5720), but also to cyclic AMP phosphodiesterase 4 (PDE4, Rolipram), and
to MEK1/2 (PD98059). The first two inhibitors test cyclic AMP signaling by
blocking protein kinase A and PDE4, respectively, The third inhibits MEK1/2,
the enzyme through which the mitogen-activated kinase cascade (i.e., Erk1/2
activation) can be stimulated by beta-adrenergic activation, an
AKAP-sensitive response (Figure 4B).
Treating the cells with any one of these specific enzyme inhibitors did not
noticeably alter AKAP12 oligomerization, i.e., the Mr
(~500 kDa) of AKAP12 on SDS-PAGE was unaffected. Finally,
we tested if the overexpression of either the N-terminal (1-938) or the
C-terminal (840-1782) fragments in HEK293 cells would affect the formation
of the SDS-resistant dimers of full-length endogenous AKAP12 (Figure 4C). The presence of neither fragment
influenced the formation of the SDS-resistant dimers (Mr
~500 kDa) of AKAP12. Both the N-terminal and C-terminal
fragments of AKAP12 expressed well in the HEK293 cells, although the
expression of the later was more robust (Figure 4C).

Figure 4

Oligomerization of AKAP12 in mammalian cells: elevation of
intracellular cyclic AMP as well as inhibition of protein kinase
A, MEK1/2, cyclic AMP phosphodiesterase 4, or expression of
either N- or C-terminal fragments do not block AKAP
oligomerization. (A) HEK293 cells were transfected with
an expression vector harboring either HA-tagged full-length AKAP12
(HA-AKAP12) or an HA-tagged AKAP12 in which alanine substitution of
protein kinase A phosphorylation sites (S627A, S696-698A, S772A) has
been performed (AKAP12M3). Cells were treated with the
beta-adrenergic agonist isoproterenol (10 μM) for 0, 5, or 30
min prior to harvesting the cells for lysis. Whole-cell lysates of
transfected cells expressing either AKAP12 or AKAP12M3 were
subjected to SDS-PAGE in the absence of 8 M urea. (B) HEK293 cells
were either untreated or incubated with a chemical inhibitor for
protein kinase A (KT5720, 1 μM), MEK1/2 (PD98059, 20
μM), or cyclic AMP phosphodiesterase 4 (Rolipram, 10 μM)
for 45 min prior to cell lysis and subsequent analysis by SDS-PAGE.
(C) HEK293 cells were either untransfected or transfected with an
expression vector harboring the AKAP12 N-terminal (1-938) fragment
or the C-terminal (840-1782) fragment. Whole-cell lysates of cells
expressing either AKAP12 or one of the fragments subjected to
SDS-PAGE in the absence of 8 M urea. The resolved proteins were
transferred to PVDF membrane, subjected to immunoblotting, and made
visible by staining with anti-HA or anti-AKAP12 antibodies.
Beta-catenin was employed as a loading control for each lane, also
identified by immunoblotting, stained with anti-beta-catenin
antibodies. The results displayed are representative of at least
three separate experiments performed on as many separate cell
cultures.

Oligomerization of AKAP5

We sought to examine if the oligomeric behavior observed for AKAP12 would be
observed in a second, much smaller, AKAP that docks to GPCRs, the AKAP5
(a.k.a., AKAP79; MW = 47.1 kDa versus 191.5 kDa for AKAP12, based upon
primary sequence). His-tagged human AKAP5 was expressed in E.
coli, purified by affinity chromatography, and subjected to to
SDS-PAGE in the absence or presence of 8 M urea (Figure 5). In the presence of either denaturant, AKAP5 behaves
as a monomer, although often as a doublet with Mr
~75 kDa (Figure 5A,
B). The microheterogenity of AKAP5 reflects, perhaps, differences
in some post-translational modifications, particularly protein
phosphorylation. The results of the protein staining were tested by
immunoblotting of the AKAP with anti-AKAP5 antibody (Figure 5C, D). The immunoblotting and protein
staining data for AKAP5 mobility on gels are in good agreement. Formation of
SDS-resistant AKAP5 oligomers, as we had observed for AKAP12, was not
observed.

Figure 5

Oligomerization of AKAP5 expressed and purified from E.
coli. His-tagged AKAP5 was expressed in
E. coli. Affinity purified AKAP5 was resolved
on either SDS-PAGE (A, C) or SDS-PAGE performed in the presence of 8
M urea (B, D). The gels were fixed and stained for protein with
Coomassie Brilliant Blue (A, B) or the resolved proteins transferred
to PVDF membrane and subjected to immunoblotting (C, D).
Immunoblotting was performed using anti-AKAP5 antibody (C, D). The
"◀" indicates the observed Mr on
the gels and the "Δ" indicates the predicted
Mr, calculated from the MW (47
kDa).

We sought to explore further the possible existence of AKAP5 dimers using
somewhat less harsh conditions. HA-tagged AKAP5 was expressed transiently in
HEK293 clones stably expressing GFP-tagged AKAP5 also. Pull-downs of
HA-tagged AKAP5 were subjected to SDS-PAGE and immunoblotting (Figure 6A). In lysates of cells expressing two
forms of exogenous AKAP5 (HA-tagged and GFP-tagged AKAP5), pull-downs of
HA-tagged AKAP5 revealed the presence of the GFP-tagged AKAP (Figure 6A). This observation clearly suggests
that AKAP5, like AKAP12, oligomerizes. The possibility of
dimerization/oligomerization of AKAP5 was interrogated also by in
vitro analysis of AKAP5-AKAP5 binding making use of c-Myc
tagged AKAP5 (expressed and purified from yeast) in combination with
His-tagged AKAP5 expressed and purified from E. coli. A
c-Myc-tagged AKAP5/anti-Myc antibody that was coupled to protein G-agarose
beads was prepared. As a control, protein G-agarose beads were similarly
coupled with mouse IgG. The purified His-tagged AKAP5 was incubated with
both types of derivatized protein G-agarose beads. Following incubation, the
agarose beads were collected, washed, and analyzed for His-tagged AKAP5
bound to the c-Myc-tagged AKAP5 (Figure 6B). AKAP5 forms oligomers. The ability of His-tagged AKAP5 to
bind immobilized AKAP5 resolves the key issue. By making use of two
different approaches with conditions less harsh that those tolerated by
AKAP12, we were able to establish that AKAP5 oligomerized. Pre-treating
cells for 45 min with enzyme inhibitors for MEK1/2 (PD98059) and PDE4
(Rolipram) had no effect on the ability of HA-tagged AKAP5 to bind
GFP-tagged AKAP5 in pull-downs of the HA-tagged version (Figure 6C), as shown by the presence of the
higher Mr GFP-tagged AKAP5 in the
precipitate.

Figure 6

Oligomerization of AKAP5 in vivo and
in vitro. (A) HEK293 cells stably
expressing GFP-tagged AKAP5 were transiently transfected with
HA-tagged AKAP5. Cell lysates were subjected to pull-downs mediated
by IgG (control) or anti-HA antibodies (IP-HA) and the pulldowns
subjected to SDS-PAGE, immunoblotting, and stained with anti-AKAP5
antibodies. Endogenous AKAP5 (expressed at <10% of that of
exogenously expressed, tagged-AKAP) display a Mr
identical with the HA-tagged version under these
conditions. (B) c-Myc-tagged AKAP5 fused to amino acids 1-147 of the
GAL4 DNA-binding domain (BD) was expressed in yeast. The c-Myc
tagged AKAP5 was subjected to pull-down with anti-c-Myc antibody or
control IgG. The immune complexes then were incubated with purified
His-tagged AKAP5. The association of His-tagged AKAP5 with the
c-Myc-tagged AKAP5 was detected by SDS-PAGE, immunoblotting, and
staining of the blots with anti-AKAP5 antibodies. (C) HEK293 cells
stably expressing GFP-tagged AKAP5 were transiently co-transfected
with an expression vector harboring HA-tagged AKAP5. The cells were
either untreated or incubated with a chemical inhibitor for either
MEK1/2 (PD98059, 20 μM) or cyclic AMP phosphodiesterase 4
(Rolipram, 10 μM) for 45 min prior to cell lysis and analysis
by SDS-PAGE. Pull-down of the HA-tagged AKAP5 was accomplished with
anti-HA antibodies. The immune precipitates were subjected to
SDS-PAGE and immunoblotting. The resolved, transferred protein blots
were stained with anti-AKAP5 antibodies. The results displayed are
representative of at least three separate experiments performed on
as many separate cell cultures.

We employed again steric-exclusion chromatography on a high-resolution,
wide-bore matrix operated by an AKTA FPLC to probe for AKAP5 oligomers
(Figure 7). His-tagged AKAP5 was
expressed and purified from E. coli. The purified AKAP5 was
subjected to steric-exclusion chromatography on Sephacryl-S400. The
chromatograms for AKAP5 and for A280 absorbance are displayed.
Immunoblotting revealed a peak displaying a Mr
of ~220 kDa and a smaller second peak displaying a
Mr. of ~44kDa, the monomeric form of
AKAP5 (Figure 7). Much like the
supermolecular complex of AKAP12, the size of the oligomer of AKAP5 was
consistent with that of an AKAP5 tetramer. When the whole-cell lysates of
E. coli expressing His-tagged AKAP5 were subjected to
the same steric-exclusion chromatography, complexes of AKAP5 were found to
display higher Mr, ~740 kDa (data not shown).
Thus, the physical properties of purified AKAP5 include the ability to form
homo-oligomeric, supermolecular complexes with Mr
suggestive of tetrameric organization.

Figure 7

Oligomerization of AKAP5 expressed and purified from E.
coil: analysis of higher-order assembly by
steric-exclusion chromatography. His-tagged AKAP5 was
expressed in E. coli. The AKAP5 was purified and
then resolved by steric-exclusion chromatography performed on an
AKTA FPLC fitted with HiPrep Sephacryl-S400 (16/60) column. The
presence of supermolecular oligomers of AKAP5 was established by
SDS-PAGE and immunoblotting of samples from the chromatography. The
resolved, transferred proteins were stained with anti-AKAP5
antibodies. Marker protein mobilities were employed to establish the
Mr of AKAP5 by elution position
(inset). The results displayed are representative of two separate
experiments performed on as many separate cell cultures.

Discussion

The current work reports on new and important properties of AKAP. First, AKAP5
and AKAP12 are shown by several analytical methods to form homodimers. The
homodimers of AKAP12 are so avidly bound that only in the presence of the strong
chaotropic agent 8 M urea are the dimers dissociated.

Oligomers of AKAP5, less avidly bound to each other, were revealed through
co-immunoprecipitation. AKAP5 is one-fourth the mass of AKAP12. Establishing the
structural basis for the oligomerization of each scaffold will require
additional studies. Second, AKAP5 and AKAP12 appear to form higher-order
oligomers. Based upon steric-exclusion chromatography, the Mr of the AKAP5
oligomers is ~220 kDa, whereas that of the AKAP12 supermolecular complexes is
~840 kDa. The presence of higher-order oligomers for two GPCR-associating AKAPs
suggests new and exciting possibilities for AKAP biology. Higher order
protein-protein interactions, in which oligomers of AKAPs interact with other
signaling molecules e.g., including GPCRs, serine/threonine
protein kinases, tyrosine kinases, phosphoprotein phosphatases, cyclic AMP
phosphodiesterase(s), et al., opens new possibilities for a
fuller understanding of AKAP function. Not known is whether oligomerization of
AKAPs (i.e., AKAP5 and AKAP12) precludes or includes the possibility of
heterodimers/heterooligomers being formed between two different AKAPs. The
presence of higher-order supermolecular complexes involving more than one AKAP
would constitute a new and important dimension to AKAP biology.

The current study demonstrates for the first time that both AKAP5 and AKAP12
display the capacity to form supermolecular homo-oligomeric structures that
likely influence the localization and function of these molecular scaffolds.
Thus AKAP oligomerization adds a new dimension on how members of this class of
scaffold molecules operate in cell signaling.

c-Myc-tagged AKAP5 fused to amino acids 1-147 of the GAL4 DNA-binding domain
(BD) was expressed in yeast and pulled down from yeast lysates with
anti-c-Myc antibody. The c-Myc-tagged AKAP5/antibody complex was absorbed to
protein G-agarose beads. Protein G-agarose beads similarly treated with
mouse IgG served as a control for this step. His-tagged AKAP5 was expressed
and purified from E. coli, as above. The purified
His-tagged AKAP5 was incubated at 25°C for 1 h in PBS containing 1%
Triton X-100 and 1 mg/ml BSA in combination with protein G-agarose beads to
which either c-Myc-tagged AKAP5 or control IgG were bound. At the end of the
incubation, the protein G-agarose beads were collected and washed in PBS
containing 1% Triton X-100 three times; the beads then were collected and
heated with 30 μl Laemmli buffer at 95°C for 5 min. The
supernatant was subjected to SDS-PAGE. AKAP5 was detected by immunoblotting
of the resolved proteins using anti-AKAP5 antibody.

Steric-exclusion chromatography of AKAP supermolecular complexes

Purified His-tagged AKAP5 or AKAP12 was applied to the Sephacryl-S400 gel
filtration column (HiPrep Sephacryl S-400 High resolution 16/60, GE
healthcare) making use of a fast-performance liquid chromatography system
AKTA (GE Healthcare), pre-equilibrated with PBS supplemented with 0.01%
NaN3. Sample (1.0 ml) fractions were collected. Each fraction
was analyzed by SDS-PAGE and immunoblotting. Protein was detected in the
flow-cell by measuring absorbance at A280.

Immunoprecipitation and immunoblotting

Cells were harvested in a lysis buffer containing 20 mM Tris-HCl, pH 7.4, 1%
Nonidet P-40, 2 mM sodium orthovanadate, 150 mM NaCl, 5 mM EDTA, 50 mM NaF,
40 mM sodium pyrophosphate, 50 mM KH2PO4, 10 mM sodium
molybdate, and a cocktail of protease inhibitors (Complete Protease
Inhibitor Cocktail tablet, Roche, Nutley, NJ). After centrifugation at
10,000 × g for 15 min, the protein concentration of
the supernatant was determined using the Bradford reagent. One mg of protein
was incubated with specific primary antibody for 4 h at 4°C, then 20
μl of protein A/G agarose was added and the mixture was incubated for
2 h on a rolling mixer. Immune complexes were collected and washed three
times with PBS containing 1% Triton X-100, and thereafter boiled for 5 min
at 95°C in 30 μl of Laemmli buffer. The supernatant was subjected
to SDS-PAGE and the resolved proteins were transferred to a PVDF membrane.
The blots were probed with specific antibodies against target proteins. The
immune complexes on the blots were made visible by staining with a
horseradish peroxidase-conjugated secondary antibody in combination with the
chemiluminescence reagent, followed by a brief autoradiography using
autoradiography film (WorldWide Medical products Inc, Hamilton, NJ).

SG collected the data and wrote the draft manuscript; HYW and CCM edited the
draft manuscript and figures of the final version of this unpublished work. Each
author read and approved the final manuscript.

Acknowledgements

The authors acknowledge the generous support by United States Public Health
Services grants DK25410-31 and DK30111-28 from the NIDDK (to C.C.M.), the
National Institutes of Health.